Previous methods for measuring plasma glycerol specific activity (SA) are sub-optimal, making the determination of glycerol kinetics in vivo with radio-tracers difficult. A new high performance liquid chromatography (HPLC) method is described which permits the accurate and specific measurement of glycerol SA. The method involves isolation of glycerol from plasma and the formation of a tribenzoyl derivative. Glycerol rate of appearance was measured in five human volunteers using both [2-3H] glycerol and [2H5] glycerol. There was close agreement between the glycerol appearance rates measured using the two approaches (1.66(0.14 vs 1.70(0.10 (mol.kg-1.min-1, respectively, P=NS). This HPLC method offers improved specificity over existing methods of measuring glycerol turnover using radio-tracers.